Background: Mitotane is the only approved treatment for patients with adrenocortical carcinoma (ACC). A better explanation for the variability in the pharmacokinetics (PK) of mitotane, and the optimization and individualization of mitotane treatment, is desirable for patients. Objectives: This study aims to develop a population PK (PopPK) model to characterize and predict the PK profiles of mitotane in patients with ACC, as well as to explore the effect of genetic variation on mitotane clearance. Ultimately, we aimed to facilitate mitotane dose optimization and individualization for patients with ACC. Methods: Mitotane concentration and dosing data were collected retrospectively from the medical records of patients with ACC taking mitotane orally and participating in the Dutch Adrenal Network. PopPK modelling analysis was performed using NONMEM (version 7.4.1). Genotypes of drug enzymes and transporters, patient demographic information, and clinical characteristics were investigated as covariates. Subsequently, simulations were performed for optimizing treatment regimens. Results: A two-compartment model with first-order absorption and elimination best described the PK data of mitotane collected from 48 patients. Lean body weight (LBW) and genotypes of CYP2C19*2 (rs4244285), SLCO1B3 699A>G (rs7311358) and SLCO1B1 571T>C (rs4149057) were found to significantly affect mitotane clearance (CL/F), which decreased the coefficient of variation (CV%) of the random inter-individual variability of CL/F from 67.0 to 43.0%. Fat amount (i.e. body weight − LBW) was found to significantly affect the central distribution volume. Simulation results indicated that determining the starting dose using the developed model is beneficial in terms of shortening the period to reach the therapeutic target and limit the risk of toxicity. A regimen that can effectively maintain mitotane concentration within 14–20 mg/L was established. Conclusions: A two-compartment PopPK model well-characterized mitotane PK profiles in patients with ACC. The CYP2C19 enzyme and SLCO1B1 and SLCO1B3 transporters may play roles in mitotane disposition. The developed model is beneficial in terms of optimizing mitotane treatment schedules and individualizing the initial dose for patients with ACC. Further validation of these findings is still required.